1. Field of the Invention
The present invention pertains to a pharmaceutically active mixture of primary high molecular weight aliphatic alcohols having enhanced purity which is isolated from beeswax. More particularly the invention pertains to a highly pure mixture of primary aliphatic alcohols which are naturally obtained from beeswax by liquid extraction from the solid wax without saponification wherein the alcohols in the mixture contain 24 to 34 carbon atoms. The C.sub.24 -C.sub.34 alcohols in the mixture advantageously consist of straight chain alcohols having 24, 26, 27, 28, 29, 30, 32 and 34 (i.e., tetracosanol, hexacosanol, heptacosanol, octacosanol, nonacosanol, triacontanol, dotriacontanol and tetratriacontanol). The invention also pertains to the method of extracting the aforementioned mixture from selected beeswax by a solid-liquid extraction procedure without saponification. The invention also pertains to the pharmaceutical use of the mixture and pharmaceutical compositions, foodstuffs and dietary supplements for administering the composition.
2. Background Information
All kinds of waxes, and more especially beeswax, have always been a matter of interest. This has been the case not only because of their industrial application, but also because of their chemical composition. The amount of beeswax in honey ranges between 0.9% to 1.13%, depending on the methods used to separate the wax from the honey. This wax is made up of monesters, hydrocarbons, free fatty acids and free alcohols.
The natural mixture of straight chain aliphatic alcohols obtained from beeswax has been studied by several authors to learn about its composition and main features. The obtaining of different groups of mixtures from all kinds of waxes has been reported in previous studies. (J. A. Lamberton et al., 1959, Australian Journal of Chemistry 13,261-268 and A. Horn and J. S. Martic, 1957 Journal of Science Food and Agriculture 10,571) and (Kreger, 1948; Wimbero, 1904; Mitsui and Col 1842). These studies suggest a method for obtaining fatty alcohols based on the homogeneous saponification with alcoholic potassium hydroxide, followed by the esterification of the unsaponifiable material and further molecular distillation.
Another method also reported is extraction of the natural alcohol mixture through a high efficiency high vacuum. The high vacuum wax distillation for the chemical isolation of carbonylic mixture derivatives and the extraction of the remaining wax is done by using petrol ether. The solvent evaporates and the remaining content is acetylated for its further isolation through alumina chromatography. Finally, through alkaline hydrolysis, alcohols are obtained and then recrystallized in ethanol, showing a fusion point ranging from 79 to 83 degrees Celsius.
Blood-lipid lowering effects of a natural mixture of straight chain aliphatic alcohols have been demonstrated by several authors (F. Liu, 1996 Active Constituents lowering blood-lipid in bees wax; Chung Kor. Chung Yao Tsa Chih 21 (9) 553-4, 576); (H. Sho et al. 1984, Effects of Okinawa sugar cane wax and fatty alcohols on serum and liver lipids in the rats; J. Nutri Vitaminol 30(6) 553-559); (S. Kato, K. Hamatani et al., 1995, Octacosanol Effects lipid metabolism in rat fed on a high fat diet; Br J Nutr 73(3) 433-441); (Kabiry et al. 1995, Tissue distribution of 8-14c) octacosanol in liver and muscle of rats after serial administration; Ann Nutr Metab 39(5) 279-284). Many investigational studies based on clinical studies with the use of the natural mixture of straight chain aliphatic alcohols have been published.
These studies have demonstrated the characteristics associated with ergogenic effects in humans and animals as well as benefits in the cardiovascular, cerebral and muscular systems. Other studies have reported that these alcohols also stimulate growth in plants-(V. Natarajan, H. H. Schmid 1997 1 -Docosanol and Other long chain primary alcohols in developing rat brain, Lipids 12(1) 128-130) (M. Azzouz, J. Borg, 1996, Enhancement of mouse sciatic nerve regeneration by the long chain fatty alcohol, N-hexacosanol, Exp Neurol 138(2) 189-197) (J. Borg, 1991 The neurotrophic Factor, n-hexacosanol, reduces the neuronal damage induced by the neurotoxin, kainic acid; J Neurosci Res (29)(1) 62-67) (J. Borg, P. J. Kesslak, C. W. Cotman, 1990, Peripheral administration of a long chain fatty alcohol promotes septal cholinergic neurons survival after fimbria fornix transection; 4; 518 (1-2)295-298) (Y. Kabir, S. Kimura 1994, Distribution of radioactive octacosanol in response to exercise in rats; 38 (4) 373-377) (R. P. Warren, R. A. Burger, R. W. Sidwell, L. L. Clark, 1992, Effect of triacontanol on numbers and functions of cells involved in inflammatory responses, 200(3)349-352) P. W. Westerman, J. M. Pope, N. Phonphok, J. W. Dan, D. W. Dubro, Biochim Biophys Acta (NETHERLANDS) 939, 64-78 (1988). Studies have been conducted regarding the partitioning of long-chain alcohols into lipid bilayers. In U.S. Pat. No. 3,031,376, Ezra Levin reported that tetracosanol, hexacosanol, octacosanol and triacontanol and their esters improved physical performance of athletes and disclosed compositions comprising such alcohols and esters in vegetable oil bases for oral ingestion. Various constituents of beeswax and products derived from beeswax have also been used in cosmetic and therapeutic applications, as disclosed by Karen M. Slimak in U.S. Pat. No. 4,793,991 which describes a hypoallergenic cosmetic comprising single plant source beeswax. Gans et al. have described the use of the non-polar saturated straight chain C.sub.21 to C.sub.33 hydrocarbon fraction of beeswax in the treatment of inflammatory skin disorders in U.S. Pat. No. 4,623,667.
A procedure for obtaining a natural mixture of straight chain higher aliphatic primary alcohols from animal and vegetable wax (a natural source wax) is also known in the prior art. This prior art procedure is based on the extraction of alcohol mixtures with fluid extractant in the sub and supercritical states between 20 and 100 degrees Celsius. Selective extraction can be carried out with this procedure but when this is applied to beeswax it is only possible to obtain 7% of C.sub.24 to C.sub.34 alcohol mixture.
Other projects (S. Inaa, K. Furukama, T. Masui, K. Honda, J. Ogasawara, and G. Tsubikamoto, 1986; Process for recovering primary normal aliphatic higher alcohols JP 60-119514) proposed a very similar extraction method applied to waxes that is based on fluids (CO.sub.2 with ethylene) in sub and supercritical states.
There are different commercial dietary supplements, foods and drugs to aid in the lowering of total blood cholesterol (lowering lipid, LDL and cholesterol levels) which are considered as effective, safe and well tolerated but most of them produce different adverse side effects. Since lipid-lowering therapy must be chronically administered, safety and tolerableness are very important for their definitive acceptance. Although many products from different sources exist in the market such as Sitosterol, garlic, bile acid binders, fibric acid derivatives, HMG-Co A reductos and Nictoinic acid, etc., the methods of use and the quantities necessary of these products are not sufficiently effective for the reduction of cholesterol to the desired levels. In addition, the drugs that are used for the lowering of cholesterol have several adverse side effects.
It has been described that treatment with some lipid-lowering drugs reduces the tendency for platelet hyperaggregation frequently seen in the hyperlipidemic patients and experimental data has shown anti-aggregatory effects mediated by these compounds. Nevertheless, only some cholesterol-lowering drugs show this property. Atherosclerosis is a variable combination of changes of the intima of the arteries consisting of the focal accumulation of lipids, complex carbohydrates, blood and blood products, fibrous tissue and calcium deposits, frequently also associated with medial changes. Thus, atherosclerosis is known as multifactorial process and includes hyperlipidemia as a risk factor.
Among the factors contributing to atherosclerosis development, platelet aggregation has a very important place. Platelet releasing granule contents activate arachidonic acid, which metabolizes into cyclic endoperoxides. These are mainly transformed into cyclic endoperoxides and finally rendering thromboxane A2 (TxA2), a strong vascular vasoconstrictor and platelet aggregatory agent. Platelet aggregation can be elicited by numerous compounds, such as collagen, ADP and epinephrine. Thus, different experimental "in vivo", "ex vivo", or "in vitro" models testing effectiveness of putative antiplatelet drugs commonly test their effect on platelet aggregation induced by these agents.
These tests are also used for testing platelet aggregation in healthy volunteers and in patients with disease which induces hyperaggregability such as hypercholesterolemia and diabetes. Collagen-induced platelet aggregation is one of the most frequently used tests. Thus, for example, collagen injected endovenously leads to reversible intravascular platelet aggregation "in vivo" and aggregates of platelet enter the vascular microcirculation, subsequently decreasing the count of circulating platelet and simultaneously increasing the plasma MDA concentration. Moreover, in some species this injection of collagen induces mortality produced by thrombosis. In these models, antiplatelet drugs generally prevent the decrease platelet content and increase of MDA concentration, as well as collagen induced mortality.
Some drugs showing platelet anti-aggregatory effects are useful for treatment of thrombotic diseases, myocardial infarction and stroke, but not all show these advantages. On the other hand, there are antithrombotic drugs such as estreptokinase and urokinase that mainly act by lytic processes affecting blood coagulation, but not on the platelet aggregation.
Since ischemic cardiovascular diseases, stroke and vascular peripheric obstructive pathologies are the main sequelae of atherosclerosis, effects of several drugs on these complications are commonly tested. Thus, theoretically a drug showing cholesterol lowering properties that also can prevent these complications by acting on other events involved in these processes must be advantageous for treating these patients. Likewise, reduction of TxA2 levels have been associated not only with antiplatelet and antithrombotic effects, but also with antischemic effects.
The pharmacological screening of antischemic drugs commonly includes the evaluation of their effects on brain-induced global ischemia. Thus, the protective effect of different drugs on rat cerebral ischemia has been determined by this type of evaluation for certain non-steroidal anti-inflammatory drugs (NSAID) which inhibits reactions catalyzed by cyclooxygenase, as well as for specific inhibitors of thromboxane synthetase and prostacyclin (PgI.sub.2) analogues(M. G. Borzeix and J. Cahn, 1988; Effects of new chemically metabolically stable prostacyclin analogues on early consequences of a transient cerebral oligemia in rats; Prostaglandins 35,5, 653-664). Other experimental models, such as global ischemia induced experimentally in Mongolian gerbils are also used frequently.
Acetylsalicylic acid (ASA) is a compound exhibiting antiplatelet, antithrombotic and antischemic properties in experimental models and human beings. It is the drug most widely used for treatment of acute myocardial infarction and stroke as well as for prevention of thromboembolic disorders. ASA effects are supported by its well known inhibition of cyclooxygenase, a key enzyme on the arachidonic acid metabolism. Thus, ASA induces a significant and remarkable reduction of serum levels of thromboxane A2 (TxA2) a recognized pathophysiological agent for the vascular endothelium and it explains the aforementioned effects of ASA.
U.S. Pat. No. 5,633,156 describes a mixture of C.sub.24 -C.sub.34 higher primary aliphatic alcohols obtained from sugar cane wax. The alcohol mixture obtained from sugar cane wax (referred to the '156 patent as MHPAA) has the same pharmaceutical utility as the beeswax mixture of higher primary aliphatic alcohols of the present invention (referred to hereinafter as BMHPAA) and can therefore be used and administered in the same manner as described in the '156 patent. However the MHPAA of the '156 patent can only be obtained by saponification of the sugar cane wax (an ester) with harsh alkali in order to obtain sufficient alcohol because sugar cane wax does not contain sufficient free alcohol to obtain a sufficient recovery of the desired alcohol product. Thus the products of saponification (i.e., alkali metal salts of the high molecular weight carboxylic acids) are formed in the material from which the alcohols must be isolated and recovered. The presence of such salts obviously poses an obstacle to the purification and recovery of the desired C.sub.24 -C.sub.34 alcohols. Furthermore, alcohols obtained from sugar cane wax, being formed by means of a chemical reaction (saponification) are not naturally derived from the wax. The term "naturally derived alcohols" as used in the present invention therefore means that the alcohols are recovered and isolated from the wax without chemical reaction with a precursor compound. In the '156 patent the alcohols are obtained by saponification of the ester precursor compounds which are naturally contained in the wax.